Supporting Information Nano-brake Halts Mitochondrial Dysfunction Cascade to Alleviate Neuropathology and Rescue Alzheimer's Cognitive Deficits

The study addresses the need for effective brain delivery of therapeutics to halt mitochondrial dysfunction in Alzheimer’s disease (AD), a critical driver of neurodegeneration. The authors hypothesize that a targeted nanocarrier delivering siRNA against cyclophilin D (CypD) can interrupt the mitochondrial dysfunction cascade, reduce neuropathology, and improve cognitive outcomes in AD. Given elevated MMP9 in damaged cerebral microvasculature of AD model mice, the team designed a matrix metalloproteinase-responsive formulation (“Nano-brake”) to enhance brain entry and intracerebral delivery specifically in AD pathology.

The supporting information cites prior work from the group and others on BBB-targeting nanocarriers and imaging-guided brain delivery, indicating a foundation in peptide-guided transport and responsive nanomaterials for CNS delivery. However, an extensive literature review is not presented in this document.

• Nanocarrier preparation: A magnesium phosphate core encapsulating CypD siRNA (Mg–siRNA core) was synthesized via a reverse water-in-oil microemulsion (Igepal CO-520/cyclohexane). Components included 50 μL of 50 μM siRNA, 300 μL of 0.625 M MgCl2 (magnesium phase), and 300 μL of 12.5 mM Na2HPO4 with 50 μL of 50 μM siRNA (phosphate phase). DOPA (20 mM, 100 μL) in chloroform was added to the phosphate phase. After mixing and stirring 40 min, the emulsion was broken with ethanol, centrifuged (12,000 g, 20 min), washed, and pellets dissolved in chloroform.
• Lipid coating: The Mg–siRNA core was coated with DMPC to form Mg-CypD-LNC. A lipid film (4 mg DMPC with Mg–siRNA core in chloroform) was rehydrated with saline and sonicated.
• Nano-brake formation: MAP peptide (sequence AC-FAEKFKEAVKDYFAKFWD-GSG-RRRRRRRRR-PVGLIG-EGGEGGEGG) was incubated with Mg-CypD-LNC at a DMPC:MAP weight ratio of 100:1 for 12 h at 4°C to yield Nano-brake. Controls included MAP-Mg-LNC (NC siRNA) and MAP-Ca-CypD-LNC (calcium phosphate core).
• Labeling: DiI/DiR dyes (0.5% for in vitro; 4% for in vivo) were added to DMPC for fluorescence tracking. For quantitative brain pharmacokinetics, d9-DMPC replaced DMPC to create d9-Nano-brake/d9-Mg-CypD-LNC for LC-MS/MS.
• Characterization: Size, PDI, and zeta potential measured by DLS (Zetasizer Nano-ZS90). TEM imaging after negative staining. Encapsulation efficiency assessed using Cy3-siRNA and fluorescence measurement after lysis at 65°C.
• Stability assays: Serum stability in 10% FBS (0–8 h) by agarose gel electrophoresis; storage stability at 4°C for 0–14 days (size, zeta, PDI, remaining siRNA by gel).
• Cell studies: bEnd.3 endothelial, SH-SY5Y neuronal, and BV2 microglial cell lines used. Cellular uptake quantified with HCS after DiI-Nano-brake incubation. Toxicity tested by CCK8 at 0–300 nM siRNA equivalent (24 h). Lysosomal colocalization and intracellular distribution assessed by confocal microscopy (FAM-siRNA and DiD-carrier), with/without MMP9 pretreatment of Nano-brake.
• MMP9 cleavage: Recombinant MMP9 activated with p-aminophenylmercury acetate and CaCl2, then incubated with Nano-brake to cleave MAP; used to probe functionality and gene silencing in cells.
• Mitochondrial function assays: After Aβ1-42 oligomer exposure (various concentrations; typically 5 μM for challenge), assessed mitochondrial membrane potential (TMRM), ROS (DCFH-DA), and mitochondrial superoxide (MitoSOX) via confocal microscopy in bEnd.3, SH-SY5Y, and BV2 cells.
• Western blot: Measured CypD, TOM20, Drp1, OPA1 in cells and brain tissue; protein extraction with RIPA, SDS-PAGE, PVDF transfer, and detection by fluorescent or HRP secondary antibodies.
• In vivo imaging: Two-photon microscopy through cranial window to visualize vascular adhesion and permeation of DiI-Nano-brake in 6-month-old 5xFAD mice; excitation 900 nm.
• Biodistribution/brain entry: IVIS imaging for DiR-labeled formulations; LC-MS/MS quantification of d9-DMPC in brain homogenates (%ID/g) in 6-month-old 5xFAD and WT littermates after tail vein injection (41.3 or 82.5 μg/kg siRNA doses).
• Immunostaining: Immunofluorescence and immunohistochemistry on paraffin brain sections (4 μm) for GLUT1, Iba1, GFAP, NeuN, and Aβ; analyzed by confocal microscopy or HRP-based detection.
• In vivo treatment: Six-month-old male 5xFAD mice received daily IV dosing of saline, Nano-brake, MAP-Mg-LNC, Mg-CypD-LNC, or MAP-Ca-CypD-LNC (82.5 μg/kg siRNA) for 4 weeks; WT saline-treated littermates as controls.
• Neuroinflammation and pathology: Cortex homogenates assayed for TNF-α and IL-6 by ELISA; Aβ burden quantified by IHC in hippocampus and cortex; ATP levels measured by kit.
• Cognitive testing: Morris Water Maze (training 4 days, probe test; tracking analysis), Novel Object Recognition (habituation, familiarization, test; tracking), and Y-maze spontaneous alternation.
• Safety: H&E of heart, liver, kidney, lung, spleen; body weight monitoring; serum renal (Urea, Cr, UA) and hepatic (T-BIL, D-BIL, I-BIL, TP, ALB, GLB, A/G, ALP, ALT, AST, AST/ALT, γ-GT) biomarkers.
• Statistics: Data as mean ± SEM or SD; Student’s t-test for two groups; ANOVA with Tukey’s post hoc for ≥3 groups; significance at p < 0.05.

• Formulation and stability: Nano-brake size, zeta potential, and PDI remained stable over 14 days at 4°C; siRNA cargo was protected from degradation in serum (10% FBS) and during storage (Figure S6). Optimization of MAP content affected particle size and charge (Figure S2).
• Cellular behavior: Nano-brake showed efficient uptake in bEnd.3 cells and limited lysosomal colocalization (Figure S3). Intracellular distribution of FAM-siRNA and DiD-carrier indicated effective delivery, influenced by MMP9 pretreatment (Figure S4). Nano-brake reduced CypD expression in BV2 and SH-SY5Y cells after MMP9 pretreatment (Western blot; n=3; p < 0.05 or p < 0.01 vs DMEM control; Figure S7). Cytotoxicity was minimal across 0–300 nM siRNA equivalent in bEnd.3 and SH-SY5Y (Figure S5).
• Brain targeting and biodistribution: Two-photon imaging showed strong adhesion and permeation of DiI-Nano-brake along cerebral vessels in 5xFAD mice between 40–70 min post-injection (Video S1). Brain entry efficiency of DiR-Nano-brake was increased in 5xFAD vs controls and higher than Mg-CypD-LNC (n=3–4; Figure S10). DiR-Nano-brake and Mg-CypD-LNC had similar peripheral organ distribution (n=4; Figure S11). DiI-Mg-CypD-LNC did not target damaged cerebral microvasculature or achieve intracerebral delivery in AD mice (Figure S9). LC-MS/MS quantified d9-DMPC in brain as %ID/g (method described).
• Disease-relevant targets: MMP9 expression was elevated in cerebral vessels of 6-month-old 5xFAD mice (Figure S8), supporting the MMP9-responsive design.
• Aβ-induced mitochondrial dysfunction in vitro: Aβ1-42 oligomers decreased mitochondrial membrane potential (TMRM) and increased ROS (DCFH-DA) in bEnd.3 cells in a concentration-dependent manner (n≈3–5; p values up to ****p < 0.0001; Figures S12, S13).
• Therapeutic effects in vitro: In SH-SY5Y and BV2 cells challenged with Aβ1-42 (5 μM, 48 h), Nano-brake alleviated mitochondrial dysfunction, reducing mitochondrial superoxide (MitoSOX) and restoring membrane potential (TMRM), outperforming Mg-CypD-LNC, MAP-Mg-LNC, and MAP-Ca-CypD-LNC (n=3–5; Figures S15, S16/S17).
• In vivo neuropathology and inflammation: Daily IV Nano-brake (82.5 μg/kg siRNA, 4 weeks) reduced cortical IL-6 and TNF-α in 5xFAD mice compared to saline (n=3–5; *p < 0.05 to ***p < 0.001; Figure S17 continuation). Nano-brake decreased Aβ deposition in hippocampus and cortex (n=4; *p < 0.05, **p < 0.01 vs saline; Figure S18).
• Behavior and safety: No change in swimming speed across groups during MWM (n=9; Figure S19), indicating motor function unaffected by treatments. H&E histology of major organs showed no overt toxicity; body weight stable (n=9). Renal (Urea, Cr, UA) and hepatic panels (T-BIL, D-BIL, I-BIL, TP, ALB, GLB, A/G, ALP, ALT, AST, AST/ALT, γ-GT) were within normal ranges across treatments (n=6–7; Figure S20).

The data support that an MMP9-responsive, magnesium phosphate core lipid nanocarrier can preferentially interact with damaged cerebral vasculature in AD, enhancing brain entry specifically in 5xFAD mice. By delivering CypD siRNA, the Nano-brake downregulated CypD, a regulator of mitochondrial permeability transition, thereby mitigating mitochondrial ROS and restoring membrane potential in Aβ-challenged neuronal and microglial cells. In vivo, chronic treatment reduced neuroinflammation (IL-6, TNF-α) and amyloid burden in key brain regions, indicating that interrupting mitochondrial dysfunction can attenuate AD-like neuropathology. Standard Mg-CypD-LNC lacking the MAP component failed to target the damaged microvasculature and did not achieve comparable brain delivery, underscoring the importance of the MMP9-responsive design. Safety assessments and unchanged swimming speeds suggest the regimen is well tolerated without affecting gross motor function. Collectively, these findings align with the hypothesis that targeted CypD knockdown can halt downstream mitochondrial damage and ameliorate AD pathology.

This work presents Nano-brake, an MMP9-responsive lipid–magnesium phosphate nanocarrier delivering CypD siRNA, which demonstrates enhanced brain entry in AD model mice, effective mitochondrial protection in vitro, and reduced neuroinflammation and amyloid deposition in vivo with favorable safety. The approach addresses key barriers in CNS delivery and targets a pivotal mitochondrial regulator. Future work should quantify long-term cognitive benefits, optimize dosing and administration schedules, investigate pharmacokinetics/pharmacodynamics in larger cohorts, and evaluate translatability in additional AD models and ultimately in nonhuman primates.

• The document is supporting information and does not provide full datasets for cognitive outcomes; behavioral improvement is inferred from the main article.
• Preclinical results are limited to one AD mouse model (5xFAD) with relatively small sample sizes in some assays (n≈3–5), which may affect generalizability.
• While MMP9 elevation supports targeting, off-target effects and detailed biodistribution kinetics beyond brain and major organs require further study.
• Mechanistic depth beyond CypD knockdown (e.g., effects on fission/fusion proteins TOM20, Drp1, OPA1) is referenced but not fully elaborated here.